INTRODUCTION

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Helicobacter pylori is a curved gram-negative bacterium that has been implicated as a major cause of chronic gastritis, peptic and duodenal ulcers, and gastric carcinoma. Its genome has been sequenced from two separate isolates revealing substantative conservation of gene sequence (2, 21). One of the most striking features of these genomes is a large (32-member) family of sequence-related outer membrane proteins. This family of proteins was discovered in the pregenomic era as a series of five outer membrane proteins named HopA to HopE, which had similar N-terminal sequences, and all reconstituted channels in planar bilayer membranes (8, 9). HopE (8) was the smallest of these proteins (31,000 Da) but formed the largest channels with a single channel conductance of 1.5 nS in 1 M KCl. For this reason it was proposed to be the major nonspecific porin of the H. pylori outer membrane, although it had a considerably lower abundance in the outer membrane than, for example, the major porin of Escherichia coli (OmpF).

HopE is also one of the smallest members of the conserved family of outer membrane proteins (2, 21; R. A. Alm, J. Bina, B. M. Andrews, P. Doig, R. E. W. Hancock, and T. J. Trust, submitted for publication). Although HopA to HopE were identified on the basis of their similar N-terminal sequences (8, 9), they have extensive blocks of C-terminal sequence conservation and in fact only 21 members of the family have the N-terminal Hop motif, and 3 of these are probably not expressed due to slipped strand regulation caused by multiple CT repeats. Even these 21 proteins can be somewhat subdivided, with 11 including HopA and HopD (10; Alm et al., submitted) and the two adhesins BabA and BabB (16) being very highly conserved and having a C terminus comprising FAY (one-letter amino acid code), whereas the remaining 9, including HopB, -C, and -E, have an F residue at the C terminus, like most other -barrel porin proteins. The 11 remaining members of the large outer membrane family do not have the typical N-terminal motif but do contain the C-terminal conserved motifs (ending in F) and have been called the Hor (Hop-related) family (Alm et al., submitted).

Overall, these 32 Hop and Hor family proteins vary substantially in size from 165 to 1,217 residues, but all contain ca. 135 to 150 conserved residues. Thus, it is of some interest to determine why these conserved residues exist. One possibility would be that the conserved sequences are required to promote homologous recombination as a mechanism for creating genomic rearrangements (21). We have previously argued against this possibility (10). Another possibility is that these sequences represent a conserved structural motif. Thus, we tested this second hypothesis here by mapping the membrane topology of HopE.

HopE, like other porins, is predicted to be a -barrel structure like, e.g., the E. coli OmpF porin. The crystal structure of OmpF (and other bacterial porins) has been determined, and it was observed that it contains 16 -strands with the general motif of alternating hydrophobic and hydrophilic residues. Interestingly, Tomb et al. (21) observed that the entire Hop and Hor families contained such alternating residues in their conserved sequences. Despite these conserved motifs, there is in fact little sequence conservation between bacterial species. Although within a species, greater conservation exists (e.g., OmpF, OmpC, and PhoE in E. coli are 80% identical), such minifamilies tend to be very similar in size (cf. the Hop and Hor family proteins). Also, the least-conserved regions within a species tend to be the surface loop regions (possibly due to antigenic selection) that interconnect each pair of -strands. This latter property has been exploited to map the membrane topology of porins (1, 3), since the surface loops can tolerate the insertion or deletion of additional amino acid residues, whereas insertions into the -strands prevent correct synthesis and secretion to the outer membrane of the mutant porin. The validity of this method has been proven by comparison to the crystal structure of E. coli porin PhoE (1, 4). Therefore, we chose this method for examining the membrane topology of HopE.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. E. coli JM105 [F' traD36 lacIQ (lacZ)M15 proA⁺B⁺] thi rpsL endA sbcB15 hsdR-4(r_K m_K⁺) (lac-proAB) was obtained from New England Biolabs, Inc. (Mississauga, Ontario, Canada) and used as the host for transformations and also for the background strain for the expression of plasmids encoding the modified HopE proteins unless otherwise stated. The plasmid pBluescript II KS(+) was obtained from PDI Bioscience (Aurora, Ontario, Canada). H. pylori 22695 (21) was obtained from the TIGR Institute of Research (Rockville, Md.).

Development of plasmid pJ1. The HopE gene was amplified from H. pylori 22695 by using Taq DNA polymerase. The upstream primer 5'-AAG GAT CCG ATA GGA ATG TAA AGG AAT GG-3' containing a BamHI site and the downstream primer 5'-CCG AAT TCT AAA GGC ATG AAC GCT TGC A-3' containing a EcoRI site were constructed by using a Perkin-Elmer Applied Biosystems, Inc. (ABI; Mississauga, Ontario, Canada) DNA synthesizer model 332. The resulting PCR fragment was blunt-end cloned into the EcoRV site in pBluescript II KS(+) in the same orientation as the lac promoter to give plasmid pJ1. All enzymes were purchased from Life Technologies-Gibco BRL. A MJ Research Minicycler (Boston, Mass.) was used for all PCR reactions. Plasmid pJ2 was derived in a similar fashion, assuming that the second Met codon at position +10 in the coding sequence was the start of the HopE gene.

Linker insertion mutagenesis. PCR primers were designed to insert the in-frame codons for five amino acids (RSKDV) and two unique restriction enzyme sites into the hopE gene, using pJ1 as the template DNA. The PCR amplification was performed with Taq DNA polymerase using a touchdown amplification procedure as follows. The PCR thermocycler was programmed for an initial denaturation step of 96°C for 4 min, followed by 18 cycles at an initial annealing temperature of 65°C (for 90 s), which was decreased by 0.5°C for each successive cycle, an extension step at 72°C for 6 min, and denaturation at 96°C for 1 min. Subsequent to completion of the first 18 cycles, an additional 14 amplification cycles were performed by using 72°C extension and 96°C denaturation steps with a constant 55°C annealing temperature. The resulting amplicon was extracted with phenol and chloroform, precipitated with ethanol, and made blunt by digestion with the Klenow fragment of DNA polymerase. The PCR products were digested with DpnI restriction enzyme to remove the template DNA, religated, and transformed into E. coli JM105. Recombinant clones were identified by using oligonucleotide primer 5'-AGA TCT AAG GAC GTC-3' plus the reverse sequencing primer in PCR amplification reactions. Identified clones were sequenced to verify that the inserted amino acids were in frame and that no errors had been introduced into the hopE gene.

Isolation of outer membrane proteins. H. pylori was grown at 37°C in an atmosphere of 10% CO₂ on chocolate agar plates (Prepared Media Laboratories, Richmond, British Columbia, Canada) overlaid with brain heart infusion broth (Accumedia, Baltimore, Md.). After an incubation period of 4 days cells were harvested from 20 plates and resuspended in 20% sucrose with 50 mg of DNase I (Boehringer Mannheim) in 10 mM Tris-HCl (pH 8.0; ICN, Aurora, Ohio). The cells were disrupted with a French pressure cell at 15,000 lb/in². Broken cells were overlaid on a sucrose step gradient of 1 ml of 70% and 6 ml of 70% sucrose (Fisher Scientific, Fair Lawn, N.J.) in 10 mM Tris-HCl (pH 8.0). The outer membrane fraction was collected and pelleted at 150,000 × g, and the pellet was resuspended in 100 µl of distilled water. E. coli JM105 transformants harboring the specified plasmids were selected on Luria-Bertani (Difco Laboratories, Detroit, Mich.) solid medium containing 0.4% glucose (wt/vol) and 100 µg of ampicillin (Sigma, St. Louis, Mo.) per ml. Twofold-concentrated YT medium (20) (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) was used for liquid cultures. Ampicillin was used at a concentration of 100 µg/µl for E. coli. After the cells were grown to the logarithmic phase, IPTG (isopropyl--D-thiogalactopyranoside; Chemica Alta, Ltd., Edmonton, Alberta, Canada) was added at a final concentration of 0.1 mM, and the cell cultures were allowed to grow another 4 h before they were harvested and resuspended in 20% sucrose with 50 mg of DNase in 10 mM Tris-HCl (pH 8.0). The outer membrane fraction was isolated as described above and pelleted at 150,000 × g, and the pellet was resuspended in 50 µl of distilled water. The protein concentration was determined using the BCA Protein Assay (Pierce, Rockford, Ill.).

HopE purification. Outer membranes from 500 ml of log-phase culture were solubilized in 10 mM Tris-HCl (pH 8.0; Fisher Scientific)-3% n-octyl-polyoxyethylene (Bachem) incubated at 23°C for 1 h and centrifuged for 30 min at 173,000 × g. The pellet was resuspended in 10 mM Tris-HCl-3% n-octyl-polyoxyethylene-5 mM EDTA (pH 8.0) (Fisher Scientific), incubated at 23°C for 1 h, and centrifuged for 30 min at 173,000 × g, and the supernatant was collected. A Western immunoblot indicated the presence of HopE in the supernatant of the second solubilization step. The supernatant containing HopE was mixed with an equal volume of 0.125 M Tris-HCl (pH 6.8), 4% (wt/vol) sodium dodecyl sulfate (SDS), and 20% (vol/vol) glycerol (Fisher Scientific) and subjected to SDS-12% polyacrylamide gel electrophoresis (PAGE). The HopE band was excised from an unstained portion of the gel and eluted overnight at 4°C into 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl. The elution supernatant was run on an SDS-PAGE gel to check for purity, and a Western immunoblot was done to ensure that it was indeed HopE.

Planar lipid bilayer experiments. The basic methods used here have been reported previously (9). Membranes were made from 1.5% oxidized cholesterol in n-decane. Bilayers were painted across a 2-mm² hole in a Teflon divider separating two compartments containing 5 to 6 ml each of a bathing solution of 1 M KCl. Voltages were applied across this membrane through Calomel electrodes connected by a salt bridge, and the resultant current was boosted 10⁹- to 10¹⁰-fold by a current amplifier, monitored on a Tektronix model 7633 oscilloscope, and recorded on a Rikadenki R-01 strip chart recorder.

SDS-PAGE and Western blotting. Isolated outer membranes were loaded at a concentration of 15 µg/lane. Electrophoresis was carried out by SDS-PAGE on a discontinuous 12% polyacrylamide gel (11). Proteins were stained with Coomassie brilliant blue. For Western immunoblotting, unstained gels were electroblotted onto Immobilon-P membranes (Millipore, Bedford, Mass.). After blocking for 2 h at 23°C with 3% bovine serum albumin (BSA; Boehringer Mannheim)-0.1% Tween 20 (Sigma) in phosphate-buffered saline (PBS), the membranes were incubated with a 1/10,000 dilution of anti-HopE rabbit antiserum (a gift from Peter Doig, Astra Zenecca, Boston, Mass.; the antiserum was raised against denatured HopE) in 1% BSA-0.05% Tween 20 in PBS for 1 h at 37°C. The membranes were then washed with PBS and incubated with a 1/5,000 dilution of an alkaline phosphatase-conjugated secondary antibody (Bio-Rad, Richmond, Calif.) for 1 h at 37°C. The bound antibodies were detected with 5-bromo-4-chloro-3-indolylphosphate (Calbiochem, La Jolla, Calif.) and nitroblue tetrazolium (Sigma).

Indirect immunofluorescence. Detection of surface-exposed proteins and epitopes was accomplished by the method of Hofstra et al. (12). For E. coli, aliquots of cells (100 µl) after 4 h of IPTG induction were pelleted, washed with PBS, and incubated with a 1/100 dilution of the primary antibody in 1% BSA in PBS for 1 h at 23°C. For H. pylori, three to four colonies were taken directly from a solid agar plate and resuspended in PBS, the cells were then pelleted, washed with PBS, and incubated with a 1/100 dilution of the primary antibody in 1% BSA in PBS for 1 h at 23°C. Cells were then washed with PBS and incubated with a 1/2,000 dilution of a fluorescein isothiocyanate-conjugated secondary antibody (Boehringer Mannheim) for 1 h at 23°C. Cells were then washed with PBS, resuspended in 1% BSA-PBS, and dried on poly-L-lysine-coated slides purchased from Sigma. Fluorescence was monitored with a Zeiss microscope fitted with a halogen lamp, and filters were set for emission at 525 nm. All images were captured using the ELIPSE software (Carl Zeiss Canada, Don Mills, Ontario, Canada).

DNA sequencing. Plasmid DNA was sequenced with the ABI automated fluorescent sequencing system model 373. Sequencing reactions were performed using the ABI sequencing kit. PCR protocols provided by ABI were done on the MJ Research Minicycler (Boston, Mass.). Template DNA was prepared with QIAwell 8 Plasmid Kits (Qiagen, Mississauga, Ontario, Canada). Primers were synthesized on the ABI DNA/RNA Model 392 synthesizer.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Cloning of HopE. There are two possible translational start sites for HopE. These comprise methionine codons three residues apart in the sequence MEFMKKF. It was felt that the second methionine was the most likely start site since the inclusion of a basic residue (E [glutamate]) anywhere in the signal sequence of any secreted protein is rare, although not unprecedented. Therefore, we cloned HopE, by PCR amplification from H. pylori genomic DNA, into the plasmid pT7-7, assuming that either the first (in plasmid pJ1) or second (in plasmid pJ2) methionine was the start codon when an exogenous Shine-Dalgarno sequence was supplied. Contrary to our expectations, only amplification from the first methionine led to the production of a heat-modifiable outer membrane protein that cross-reacted with antibody raised against denatured HopE protein (Fig. 1A, cf. lanes 3 and 4 and lanes 5 and 6). Plasmid pJ1 encoded this version of HopE (with its endogenous Shine-Dalgarno sequence) cloned into pBluescript II KS(+). This construct was transformed into E. coli JM105 and induced with 0.1 mM IPTG for 4 h. Outer membranes were isolated by sucrose density gradient centrifugation and shown to contain an outer membrane protein with an apparent molecular weight of 31,000 that comigrated with authentic HopE on SDS-PAGE and reacted with antibody to HopE (Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   (A) Western immunoblot probed with anti-HopE antibodies of outer membranes of E. coli JM105 clones solubilized at 100°C (heated) or 23°C (unheated). Lanes 1 and 2, JM105/pBluescript heated and unheated; lanes 3 and 4, JM105/pJ1 heated and unheated; lanes 5 and 6, JM105/pJ2 heated and unheated; lanes 7 and 8, H. pylori heated and unheated. Approximately 20 µg of total protein per lane was loaded. The anti-HopE antibodies were raised against denatured HopE and thus reacted more strongly to heated (denatured) HopE rather than unheated HopE in which some linear epitopes were presumably buried. (B) SDS-PAGE demonstrating the purity of HopE isolated from E. coli JM105/pJ1. Lane 1, solubilized at 100°C (heated); lane 2, solubilized at 23°C (unheated).

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Conductance trace observed after the addition of 3 ng of native HopE per ml to the aqueous phase (1 M KCl) bathing a planar lipid bilayer constituted from 1.5% oxidized cholesterol in n-decane. The applied voltage was 50 mV. The arrows indicate the breakpoints for three channels that entered the membrane rapidly.

Construction of a membrane topology model. The signal sequence cleavage site for HopE is known due to its known N-terminal sequence EGDGVYIGTNY (8). The mature HopE amino acid sequence in both sequenced genomes, strain J99 (2) and 26695 (21), was very similar, with only 6 amino acids of 250 being different. We utilized the method of Jeanteur and Pattus (14, 17) to predict the transmembrane -strands of mature HopE (Fig. 3). This method utilizes a window of five amino acids to predict amphipathic regions of the protein comprising alternating hydrophobic and hydrophilic amino acids. In the crystallized porins, the alternation of hydrophobic and hydrophilic residues is incomplete (i.e., sometimes a polar residue appears in place of a hydrophobic residues and vice versa), in part because porins often form trimeric structures. Thus, the surfaces of the monomers that contact each other do not necessarily need to be hydrophobic like the surfaces that contact the membrane interior. Also, at least one of the surface loop regions interconnecting transmembrane -strands (usually loop 3) inserts into the center of the barrel, and hydrophobic residues pointing into the aqueous center of the -barrel pore can interact with these residues to form hydrophobic contacts. Thus, the averaging over a window of five residues accommodates such inconsistencies.

View larger version (27K): [in this window] [in a new window]   FIG. 3.   Membrane topology model of HopE. Amino acids that are highly conserved throughout the Hop and Hor families of outer membrane proteins are shown in italics and underlined. Tolerated insertions are indicated by the filled arrows, and nontolerated insertions are indicated by the open arrows.

Testing of the membrane topology model. Alignment of the sequenced porins has demonstrated that the -strand regions and turns tend to be quite conserved in length, whereas the surface loops are quite variable in length (17). Consistent with this idea, insertions and deletions into the surface loop regions of individual porins are usually permissible (1, 3, 17), with the exception that some insertions into loop 3 (which folds into the interior of the -barrel in the crystallized porins) disrupt the secretion and/or stability of the protein (1, 14). On the other hand, insertions or deletions in the -strands and/or turn regions of porins always perturb the secretion and/or stability of the porin (1, 3, 14). Therefore, we set about testing the membrane topology model by inserting, using PCR methodology, the in-frame sequence RSKDV. The inclusion of four consecutive polar residues, three of which are charged, into HopE -strands would be expected to disrupt this protein. A total of 12 insertions were made, with a further 2 insertions being made in which an accompanying deletion was constructed. The position of these insertions in the mature HopE sequence and their predicted locations are indicated in Table 1 and Fig. 3. The expression of HopE and its modification by heat was observed on Western immunoblots of whole-cell and outer membrane preparations (Fig. 4), and the surface expression was determined by indirect immunofluorescence with HopE-specific antibody. We considered that indirect immunofluorescence with intact cells was the best indicator of the correct localization of the HopE mutants in the E. coli outer membrane. Heat modification on SDS-PAGE is a signature property of many porins, but the loop 5 deletion mutant of porin OprD, for example, is more susceptible to denaturation in SDS (and thus does not demonstrate heat modification) (13) despite clear evidence that this mutant forms a native -barrel structure (15). Consistent with this, mutant pJ18 in predicted loop 2 and mutant pJ20 in predicted loop 8 demonstrated an apparent surface location (Table 1) but were not apparently heat modifiable (Fig. 4).

View larger version (46K): [in this window] [in a new window]   FIG. 4.   Western immunoblot probed with anti-HopE antibodies of sucrose gradients of whole-cell proteins of E. coli JM105 clones or H. pylori solubilized at 100°C (odd-numbered lanes) or 23°C (even-numbered lanes). Lanes 1 and 2, JM105/pJ20; lanes 3 and 4, JM105/pJ21; lanes 5 and 6, H. pylori OM; lanes 7 and 8, JM105/pJ34; lanes 9 and 10, JM105/pBluescript; lanes 11 and 12, JM105/pJ31; lanes 13 and 14, JM105/pJ32; lanes 15 and 16, are H. pylori. Approximately 15 µg of total protein was loaded per lane.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Histogram showing single-channel conductance measurements in 1 M KCl under the conditions described in Fig. 3 for native HopE and HopE with the loop 2 insertion.